Apolipoprotein M (apoM) is a newly characterized human apolipoprotein that is mainly associated with high-density lipoprotein (HDL) in plasma and for a small proportion with triglyceride-rich lipoproteins (TGRLP) and low-density lipoproteins (LDL). ApoM fulfills the criteria of an HDL-associated apolipoprotein since the majority of apoM in plasma is associated with HDL. In situ hybridization experiments have demonstrated that apoM is exclusively expressed in man in hepatocytes and kidney tubular cells. Previous studies have suggested that apoM may be involved in lipid and/or lipoprotein metabolism. For example, the proportion of apoM in TGRLP is increased in the postprandial phase. Recently, we reported that apoM expression is associated with activation of the platelet-activating factor receptor (PAF-R) in HepG2 cells, such that PAF enhances apoM expression and secretion in a dose-dependent manner, whereas a PAF-R antagonist, lexipafant, inhibits apoM expression. Nevertheless, the (patho)physiological functions of apoM are still not understood.

ApoM genes are found in all mammalian genomes and are highly conserved. The identity of cDNA sequences or amino acid sequences of human, rat and mouse apoM is over 80%. In the present study, we investigated apoM expression patterns during mouse and human embryogenesis using northern blotting of full-stage mouse embryos, mRNA arrays of human fetal normal tissues and in situ hybridization of sections of mouse embryos with the use of an alkaline phosphatase-conjugated antisense oligonucleotide.

Northern blots of mouse embryo (4.5–18.5 days postcoitus) were purchased from BioCat (Heidelberg, Germany). Two different batches of the blots were applied in the present study. Sections of 12 day- and 15 day-old mouse embryos were purchased from Novagen (VWR International AB, Stockholm, Sweden). mRNA arrays of normal human fetal tissues were purchased from BioChain Institute (Hayward CA, USA). An alkaline phosphatase-conjugated antisense oligonucleotide (AP-cacagaccccacttttcgtgcggatggtagcacgaa) probe of mouse apoM cDNA 380-315 was purchased from DNA-Technology (Aarhus, Denmark).

A commercial mouse GAPDH cDNA (Clontech, Palo Alto CA, USA) was used as control probe. A full-length mouse apoM cDNA was amplified by polymerase chain reaction (PCR) (primers: 5?-atgttccaccaagtttgggc and 5?-cttgctggacagcgggcaggcc) from a mouse liver cDNA library, and was used as mouse apoM probe for northern blots. PCR reactions were performed using Ampli Taq DNA polymerase with buffers and dNTPs (all from Perkin Elmer, Roche Molecular Systems, Boston, MA USA) on a GeneAmp PCR System 2400 (Perkin Elmer, Applied Biosystems, Boston, MA USA). In the mRNA arrays of normal human fetal tissues, a full-length human apoM cDNA was used as probe and a commercial human GAPDH cDNA was used as control probe. Probes were radiolabeled with [³²P]dCTP using the random primer method (RediPrime; Amersham Pharmacia Biotech, Uppsala, Sweden). Two northern blot membranes of different batches were used in the present study. Hybridizations were carried out at 65°C in a hybridization solution (Clontech, Palo Alto CA, USA). The blots were washed several times in 2×SSC (1×SSC=150 mM sodium chloride, 15 mM sodium citrate, pH 7.0) containing 0.1% SDS at room temperature for 2 h and twice in 0.1×SSC containing 0.05% SDS at 50°C for 40 min. The blots were then exposed to X-ray film at ?70°C for 24 h. Autoradiographs were analyzed with a 1600 scanner (Epson, Sollentuna, Sweden). The membranes were stripped with boiled water in the presence of 0.5% SDS for 10 min and then rehybridized with the next probe.

Commercial mouse embryo sections were subjected to in situ hybridization using an AP-conjugated apoM antisense oligonucleotide probe (AP-probe) specific for apoM mRNA. Specificity of the AP-probe was evaluated by comparing with the full-length antisense mouse apoM cDNA probe. Sections were dehydrated once in a graded series of ethanol (30%, 50%, 70%, 95% and 100%, respectively) and subsequently left to dry in air. Sections were stored at 4°C before hybridization. In brief, reactions were performed for 2 h at 37°C, the DNA templates were removed by incubation for 15 min at 37°C with 10 U RNase-free DNase I (Roche Diagnostics Scandinavia AB, Bromma, Sweden), and the reactions were terminated by adding 1/10 (v/v) of 200 mM EDTA, pH 8.0. Sections were treated at room temperature with 0.5 N HCl for 5 min to disrupt ribosomes, followed by an incubation with 1 ?g/ml proteinase K for 5 min, and then with 0.1 M triethanolamine-HCl (pH 8.0) for 5 min before hybridization. After prehybridization in 50% formamide, 5×SSC and 40 ?g/ml salmon sperm DNA at 50°C for 2 h, sections were hybridized with 0.6 ?g/ml AP-probe in 50% deionized formamide, 2.5 mM EDTA buffer (pH 8.0), 300 mM NaCl, 1× Denhardt’s solution, 10% dextran sulfate, and 1 mg/ml brewer’s yeast tRNA at 50°C overnight. After hybridization, sections were washed on a shaker in 50% deionized formamide and 2×SSC for 60 min at 50°C, and then subjected to digestion with 10 mg/ml RNase A at 37°C for 30 min. Then, sections were washed in 50% deionized formamide and 2×SSC for 60 min at 50°C, followed by washing in 1×SSC, and 50% deionized formamide at 50°C for 60 min. The hybridized AP-probe was visualized with 5-bromo-4-chloro-3-indolyl phosphate as substrate and nitroblue tetrazolium. Haematoxylin-eosin (H&E) staining was performed according to a standard procedure.

To elucidate whether and when apoM is expressed during the early stages of mouse ontogeny, northern blots of apoM using total RNA from entire mouse embryos were performed. ApoM transcripts were detected in 7.5–18.5 day-old embryos. ApoM was expressed at a low level on day 7.5, its expression increased significantly on day 9.5, markedly decreased on day 10.5, and then increased continuously up to day 18.5. Hybridization using the GAPDH cDNA probe was performed as loading control. Fig. 2 shows the organs that express apoM in sections of mouse embryos of day 12 and day 15. In 12 day-old embryos apoM was found to be mainly expressed in the liver, and in 15 day-old embryos, apoM was expressed in both liver and kidney.

Figure 1. Onset of apoM expression during mouse embryogenesis. Northern blots of total RNA of 4.5–18.5-day-old mouse embryos was hybridized to a randomly primed cDNA probe of apoM. Each lane contained 20 ?g total RNA from an entire embryo. Random priming was achieved with radio-labeled [³²P]dCTP, and a final specific activity higher than 10⁹ cpm/g DNA was obtained. Mouse GAPDH cDNA was used as control. The experiments were repeated twice with membranes of different batches.

Figure 2. In situ hybridization of mouse embryos. In situ hybridization of apoM mRNA performed with an AP-conjugated antisense oligonucleotide using sections of mouse embryos. Panel A (day 12) and panel C (day 15) show H&E stained sections. Panel B (day 12) and panel D (day 15) show apoM mRNA expression patterns. Apical is up, and dorsal is left. Original magnifications, ×1.6.

ApoM was strongly expressed in livers of 3–5 month-old human embryos and continued to be strongly expressed throughout embryogenesis. In the kidney, apoM expression was highest in 5–9 month-old embryos. There was some expression of apoM in small intestine, particularly in later stages of embryogenesis. In skeletal muscle, minute apoM expression was found in 3–5 months-old embryos, and some apoM expression was found in stomach in earlier stages of embryogenesis.